Plant characterization of insect-protected soybean.

Insect-protected soybean (SIP) that produces the Cry1A.105 and Cry2Ab2 insecticidal crystal proteins has been developed to provide protection from feeding damage caused by targeted lepidopteran insect pests. Typically, as part of environmental risk assessment (ERA), plant characterization is conducted, and the data submitted to regulatory agencies prior to commercialization of genetically modified (GM) crops. The objectives of this research were to: (a) compare soybean with and without the SIP trait in plant characterization field trials designed to fulfill requirements for submissions to global regulatory agencies and address China-specific considerations and (b) compare risk assessment conclusions across regions and the methodologies used in the field trials. The soybean with and without the SIP trait in temperate, tropical, and subtropical germplasm were planted in replicated multi-location trials in the USA (in 2012 and 2018) and Brazil (in 2013/2014 and 2017/2018). Agronomic, phenotypic, plant competitiveness, and survival characteristics were assessed for soybean entries with and without the SIP trait. Regardless of genetic background, growing region, season, or testing methodology, the risk assessment conclusions were the same: the evaluated insect-protected soybean did not differ from conventional soybean in evaluated agronomic, phenotypic, competitiveness, and survival characteristics indicating no change in plant pest/weed potential. These results reinforce the concept of data transportability across global regions, different seasons, germplasm, and methodologies that should be considered when assessing environmental risks of GM crops.

Seed production of wild soybean (Glycine soja Sieb. et Zucc.) under favorable, ruderal, and natural growing conditions.

Field trials were conducted in Japan under different growing conditions to better understand seed production of wild soybean (Glycine soja Seib. et Zucc.). The objectives of these trials were to evaluate yield and yield components of wild soybean: (1) across 11 diverse populations grown under favorable conditions to assess seed production potential, (2) under different planting densities (112, 208, 416, and 832 plants/m2) to assess intra-specific competition, and (3) across growing conditions (favorable, ruderal, and natural) to assess the impacts of environmental stress and inter-specific competition. Significant differences in yield and seed number observed among the evaluated wild soybean populations were predominantly due to environmental effects and genetic by environmental interaction. Seed production was impacted by both intra- and inter-specific competition. Wild soybean grown without plant competition had 51-fold and 247-fold higher yield compared to when grown in ruderal and natural environments, respectively. Under favorable growing conditions, wild soybean had substantial yield potential due to the ability to produce a high number of seeds. In nature, yield potential is severely limited because of plant competition and other environmental stressors. The results of this research are useful to inform environmental risk assessment when considering the potential impact of soybean biotechnology traits that increase or protect yield. If such traits were to be inadvertently transferred from imported soybean into wild soybean, this research indicates that the effects would likely have little overall impact on wild soybean seed production.

Assessment of potential impacts associated with gene flow from transgenic hybrids to Mexican maize landraces.

Genetically modified (GM) maize has been grown and safely consumed on a global scale since its commercialization in 1996. However, questions have been raised about the potential impact that GM maize could have on native maize landraces in Mexico, which is the center of origin and diversity of maize. This research was conducted to evaluate potential changes to maize landraces in an unlikely event of transgene introgression. For this study, two GM traits that confer insect protection and herbicide tolerance in maize (MON 89034 and MON 88017), designated as VT3Pro, were introgressed into two Mexican landraces, Tuxpeño and Tabloncillo. Field trials were conducted across four environments to assess phenotypic characteristics, plant response to stressors, and kernel composition of landraces with and without VT3Pro traits. Furthermore, materials from four backcrossing generations were analyzed for segregation of these GM traits. Generally, no significant differences were observed between landraces with and without VT3Pro traits for the evaluated characteristics and the segregation analysis showed that GM traits, when introgressed into landraces, followed Mendelian principles. These results support the conclusion that, if inadvertently introgressed into landraces, VT3Pro traits are not expected to alter phenotypic or kernel characteristics, plant response to stressors (except for targeted insect protection and herbicide tolerance traits) and would segregate like any endogenous gene. These results should be taken into consideration when discussing benefits and risks associated with commercial production of GM maize hybrids in the centers of origin and diversity of maize.

Variability of CP4 EPSPS expression in genetically engineered soybean (Glycine max L. Merrill).

The expression of the CP4 EPSPS protein in genetically engineered (GE) soybean confers tolerance to the Roundup® family of agricultural herbicides. This study evaluated the variability of CP4 EPSPS expression using an enzyme-linked immunosorbent assay in soybean tissues collected across diverse germplasm and 74 different environments in Argentina, Brazil and the USA. Evaluated material included single and combined (stacked) trait products with other GE traits in entries with cp4 epsps gene at one or two loci. The highest level of CP4 EPSPS was observed in leaf tissues, intermediate in forage and seed, and lowest in root tissues. Varieties with two loci had approximately twice the level of CP4 EPSPS expression compared to one locus entries. Variable and non-directional level of CP4 EPSPS was observed with other factors like genetic background, trait stacking, growing region or season. The maximum and average CP4 EPSPS expression levels in seed provided large margins of exposure (MOE of approximately 4000 and 11,000, respectively), mitigating concerns over exposure to this protein in food and feed from soybean varieties tolerant to Roundup® herbicides.

Likelihood assessment for gene flow of transgenes from imported genetically modified soybean (Glycine max (L.) Merr.) to wild soybean (Glycine soja Seib. et Zucc.) in Japan as a component of environmental risk assessment.

Environmental risk assessment is required for genetically modified (GM) crops before their import into Japan. Annual roadside monitoring along transportation routes from ports to processing facilities for GM soybean (Glycine max (L.) Merr.) have been requested as a condition of import only approval because of lack of information on the likelihood of persistence of imported GM soybean for food, feed and processing and the potential for transfer of transgenes into wild soybean (Glycine soja Seib. et Zucc.) through gene flow under the Japanese environment. The survey of soybean seeds, plants and wild soybean populations were conducted along transportation routes from unloading ports to processing facilities that provided data to help quantify actual exposure. The survey indicated that the opportunities for co-existence and subsequent crossing between wild soybean populations and imported soybean are highly unlikely. Together the survey results and the comprehensive literature review demonstrated low exposure of imported GM soybean used for food, feed and processing in Japan. This evaluation of exposure level is not specific to particular GM soybean event but can apply to any GM soybean traits used for food, feed and processing if their weediness or invasiveness are the same as those of the conventional soybean.

Assessment of the potential for gene flow from transgenic maize (Zea mays L.) to eastern gamagrass (Tripsacum dactyloides L.).

Eastern gamagrass (Tripsacum dactyloides L.) belongs to the same tribe of the Poaceae family as maize (Zea mays L.) and grows naturally in the same region where maize is commercially produced in the USA. Although no evidence exists of gene flow from maize to eastern gamagrass in nature, experimental crosses between the two species were produced using specific techniques. As part of environmental risk assessment, the possibility of transgene flow from maize to eastern gamagrass populations in nature was evaluated with the objectives: (1) to assess the seeds of eastern gamagrass populations naturally growing near commercial maize fields for the presence of a transgenic glyphosate-tolerance gene (cp4 epsps) that would indicate cross-pollination between the two species, and (2) to evaluate the possibility of interspecific hybridization between transgenic maize used as male parent and eastern gamagrass used as female parent. A total of 46,643 seeds from 54 eastern gamagrass populations collected in proximity of maize fields in Illinois, USA were planted in a field in 2014 and 2015. Emerged seedlings were treated with glyphosate herbicide and assessed for survival. An additional 48,000 seeds from the same 54 eastern gamagrass populations were tested for the presence of the cp4 epsps transgene markers using TaqMan® PCR method. The results from these trials showed that no seedlings survived the herbicide treatment and no seed indicated presence of the herbicide tolerant cp4 epsps transgene, even though these eastern gamagrass populations were exposed to glyphosate-tolerant maize pollen for years. Furthermore, no interspecific hybrid seeds were produced from 135 hand-pollination attempts involving 1529 eastern gamagrass spikelets exposed to maize pollen. Together, these results indicate that there is no evidence of gene flow from maize to eastern gamagrass in natural habitats. The outcome of this study should be taken in consideration when assessing for environmental risks regarding the consequence of gene flow from transgenic maize to its wild relatives.

Natural Variability of Allergen Levels in Conventional Soybeans: Assessing Variation across North and South America from Five Production Years.

Soybean (Glycine max L. Merrill) is one of eight major allergenic foods with endogenous proteins identified as allergens. To better understand the natural variability of five soybean allergens (Gly m 4, Gly m 5, Gly m 6, Gly m Bd 28k, and Gly m Bd 30k), validated enzyme-linked immunosorbent assays (ELISAs) were developed. These ELISAs measured allergens in 604 soybean samples collected from locations in North and South America over five growing seasons (2009-2013/2014) and including 37 conventional varieties. Levels of these five allergens varied 5-19-fold. Multivariate statistical analyses and pairwise comparisons show that environmental factors have a larger effect on allergen levels than genetic factors. Therefore, from year to year, consumers are exposed to highly variable levels of allergens in soy-based foods, bringing into question whether quantitative comparison of endogenous allergen levels of new genetically modified soybean adds meaningful information to their overall safety risk assessment.

Characterization of Natural and Simulated Herbivory on Wild Soybean (Glycine soja Seib. et Zucc.) for Use in Ecological Risk Assessment of Insect Protected Soybean.

Insect-protected soybean (Glycine max (L.) Merr.) was developed to protect against foliage feeding by certain Lepidopteran insects. The assessment of potential consequences of transgene introgression from soybean to wild soybean (Glycine soja Seib. et Zucc.) is required as one aspect of the environmental risk assessment (ERA) in Japan. A potential hazard of insect-protected soybean may be hypothesized as transfer of a trait by gene flow to wild soybean and subsequent reduction in foliage feeding by Lepidopteran insects that result in increased weediness of wild soybean in Japan. To assess this potential hazard two studies were conducted. A three-year survey of wild soybean populations in Japan was conducted to establish basic information on foliage damage caused by different herbivores. When assessed across all populations and years within each prefecture, the total foliage from different herbivores was ≤ 30%, with the lowest levels of defoliation (< 2%) caused by Lepidopteran insects. A separate experiment using five levels of simulated defoliation (0%, 10%, 25%, 50% and 100%) was conducted to assess the impact on pod and seed production and time to maturity of wild soybean. The results indicated that there was no decrease in wild soybean plants pod or seed number or time to maturity at defoliation rates up to 50%. The results from these experiments indicate that wild soybean is not limited by lepidopteran feeding and has an ability to compensate for defoliation levels observed in nature. Therefore, the potential hazard to wild soybean from the importation of insect-protected soybean for food and feed into Japan is negligible.

Pollen-Mediated Gene Flow in Maize: Implications for Isolation Requirements and Coexistence in Mexico, the Center of Origin of Maize.

Mexico, the center of origin of maize (Zea mays L.), has taken actions to preserve the identity and diversity of maize landraces and wild relatives. Historically, spatial isolation has been used in seed production to maintain seed purity. Spatial isolation can also be a key component for a strategy to minimize pollen-mediated gene flow in Mexico between transgenic maize and sexually compatible plants of maize conventional hybrids, landraces, and wild relatives. The objective of this research was to generate field maize-to-maize outcrossing data to help guide coexistence discussions in Mexico. In this study, outcrossing rates were determined and modeled from eight locations in six northern states, which represent the most economically important areas for the cultivation of hybrid maize in Mexico. At each site, pollen source plots were planted with a yellow-kernel maize hybrid and surrounded by plots with a white-kernel conventional maize hybrid (pollen recipient) of the same maturity. Outcrossing rates were then quantified by assessing the number of yellow kernels harvested from white-kernel hybrid plots. The highest outcrossing values were observed near the pollen source (12.9% at 1 m distance). The outcrossing levels declined sharply to 4.6, 2.7, 1.4, 1.0, 0.9, 0.5, and 0.5% as the distance from the pollen source increased to 2, 4, 8, 12, 16, 20, and 25 m, respectively. At distances beyond 20 m outcrossing values at all locations were below 1%. These trends are consistent with studies conducted in other world regions. The results suggest that coexistence measures that have been implemented in other geographies, such as spatial isolation, would be successful in Mexico to minimize transgenic maize pollen flow to conventional maize hybrids, landraces and wild relatives.